Project Summary/Abstract Understanding how the various neurons differentiate from neural progenitor cells is essential for understanding both the biology and diseases of the central nervous system, including the retina. All retinal cell types, including the seven types of neurons and the Mller glial cells, arise from retinal progenitor cells (RPCs) during development. Defects in any of the retinal cell types can cause vision loss and even blindness. Among the various retinal cells, retinal ganglion cells (RGCs) are the only output neurons, which send axons to the retinorecipient target regions of the brain in the form of the optic nerve. Our long-term goal is to obtain a comprehensive understanding of the genetic and molecular mechanisms regulating RGC formation during development. Three transcription factors serve as key regulators in RGC differentiation; ATOH7 functions upstream to render RPCs competent for the RGC lineage, whereas POU4F2 and ISL1 function downstream to promote RGC differentiation. In the last funding cycle, we for the first time established that POU4F2 and ISL1 are sufficient to specify the RGC fate and promote their differentiation, further clarifying and establishing the critical roles these two factors play in RGC genesis. Despite the progress we and others have made, major questions remain in our understanding of RGC development. Specifically, it remains unclear what makes ATOH7-expressing RPCs unique so that RGCs arise from them, how RGC-specific genes are activated, what specific roles ATOH7 plays in these processes, and how RGC precursors, once committed to their fate, further differentiate into mature and functional RGCs. In this proposal, we aim to address these important issues. Our overarching hypothesis is that changes in the epigenetic landscape are the major mechanism underlying the progression of RGC genesis through the different phases. Thus, investigating how the epigenetic landscape evolves to influence gene expression during RGC formation will be the central theme of this proposal. Our specific aims, which are all designed around, this theme, include: 1) to investigate the properties of RGC- competent RPCs by clonal lineage analysis, expression profiling, and epigenomics survey; 2) to survey the progression of the epigenetic landscape through RGC differentiation and identify RGC-specific enhancers in the genome; and 3) to experimentally evaluate and characterize the function of RGC-specific enhancers. Collectively, these aims address the genetic and epigenetic basis underlying RGC formation. The expected results will expand our knowledge about the molecular events during the transition from RPCs to RGCs in retinal development. They will also help us to define the requirement for reprograming stem cells into RGCs, which is highly pertinent to developing treatment for RGC-related diseases. Thus, our proposed research is significant in both advancing basic research on retinal development and providing knowledge for clinical applications.